PRINCIPLES, PRACTICE AND PRESSING QUESTIONS OF METHYLMERCURY TOXICOKINETICS AND TOXICODYNAMICS IN HUMANS AND ANIMAL MODELS
The fate of mercury in the body after an environmental exposure to methylmercury (MeHg) is dictated by fundamental principles of toxicokinetics. Understanding the potential impact of MeHg toxicity therefore requires a working knowledge of the characteristics of adsorption, distribution, metabolism and excretion. In addition, understanding MeHg toxicity at relevant sites of action, such as in the fetus and the brain, requires insight into toxicodynamic interactions with tissue specific factors and nutrients in various compartments of the body. The extent of variation in toxicokinetic traits, such as elimination rate, which dictates the MeHg half-life (t1/2), is incompletely characterized in both humans and animal models. For example, methylmercury half-life in humans is reported to range from t1/2 ~ 30 to >120 days, yet underlying factors controlling this variation are not known. Furthermore, recent evidence from our own studies demonstrates that elimination rate can vary within an individual over time. A working knowledge of toxicokinetics and toxicodynamics is even more essential for interpreting complex studies on the genetic, environmental and nutritional factors that modify methylmercury toxicity as well as for characterizing the variety of environmental sources of MeHg exposure. This presentation aims provide a condensed summary of the toxicokinetic and toxicodynamic principles specific to MeHg. Examples of representative studies of half-life determination in human and animal models will be presented to illustrate both past and emerging methodologies and to highlight examples of toxicokinetic parameters currently used for regulatory guidelines of MeHg exposure. How toxicokinetics and toxicodynamics are applicable to interpreting MeHg exposures in individuals and populations, with particular attention to fetal and early life exposures, will be outlined. Finally, gaps in understanding methylmercury toxicokinetics and toxicodynamics will be laid out to enhance the discussion of the way forward in this fundamental area of mercury research.
METHYLMERCURY VERSUS SELENIUM, VITAMIN E, AND DOCOSAHEXAENOIC ACID IN FETAL CIRCULATION: COMPARISON WITH MATERNAL STATUS
Methylmercury (MeHg) is a neurotoxicant that the developing brain of a fetus is especially susceptible to. On the other hand, selenium, vitamin E (VE), and docosahexaenoic acid (DHA) have been reported to protect MeHg toxicity. The placenta strongly controls the biochemical composition of the fetal circulation by regulating entry of nutrients derived from the mother. Therefore, the study of the characteristics of the profiles of mercury in fetal circulation as well as those of selenium, VE, and DHA, potential protective factors against the toxicity of MeHg, are important to evaluate the high susceptibility of the fetus. The objectives of this study were to: 1) investigate the characteristic of the placental transfer of nutrients and MeHg by comparing biochemical composition in the fetal and maternal blood and 2) investigate the difference of the status of Hg relative to Se, VE, and DHA in fetal blood versus maternal blood to estimate the sensitivity of the fetus to MeHg toxicity. Blood samples were collected separately from the maternal and umbilical vein at parturition from 54 mother-infant pairs of Japanese. The characteristics of fetal circulation were low contents of lipid components and fatty acids and high contents of amino acids, including methionine, compared with those in maternal circulation. Mercury in cord blood (7.26 ng/g) was higher than in maternal blood (185%). Selenium in cord blood (153 ng/g) was similar to maternal blood. On the other hand, VE (0.31 mg/dl) and DHA (57.9µg/ml) in cord blood were lower than maternal blood (45% for VE and 22% for DHA). These results showed that the ratios of selenium/mercury, DHA/mercury, and VE/mercury were lower in fetal circulation than those in maternal blood. Not only the approximately two times higher Hg but also the lower ratios of protective factors such as selenium, VE and DHA against mercury in fetal circulation may contribute to the high susceptibility of a fetus to MeHg toxicity.
BIOAVAILABILITY OF METHYLMERCURY FROM COMMONLY CONSUMED SEAFOOD: AN IN VITRO INVESTIGATION OF GASTROINTESTINAL AND COLONIC DIGESTION
Seafood provides essential nutrients, but is also contaminated with methylmercury (MeHg). Current risk assessments for ingested MeHg assumes it is 100% bioavailable in the human gastrointestinal tract. However, recent studies on MeHg bioavailability suggest otherwise. These studies were mostly performed outside of North America, on seafood not commonly consumed in North America, and focus almost exclusively on bioaccessibility (digestive processes) rather than bioavailability (cumulative digestive + absorptive processes). We aimed to evaluate both MeHg bioaccessibility and bioavailability from edible tissue of the top 10 most consumed types of seafood in the United States, identified by a previous survey; as well as assess the role of the colon in MeHg bioaccessibility and bioavailability. We used an in vitro model of human digestion including gastric and small intestinal phases; some digestions also included a colonic phase. Bioaccessible MeHg was measured in the soluble fraction resulting from these digestions. Soluble fraction was added to a Caco-2 Transwell assay to assess MeHg bioavailability. After gastrointestinal digestion, mean MeHg concentrations ranged from 0.73 ng/g (scallop) to 527 ng/g (canned white tuna) in undigested seafood, from 0.73 ng/g (scallop) to 366 ng/g (fresh tuna) in bioaccessible fraction, and from 0.34 ng/g (scallop) to 266 ng/g (fresh tuna) in bioavailable fraction. MeHg bioaccessibility ranged from 50% (canned white tuna) to 100% (shrimp, scallop), and bioavailability from 29% (crab) to 67% (salmon). In preliminary studies, colonic digestion of fresh tuna reduced MeHg bioaccessibility by 88% relative to gastrointestinal digestion only; colonic digestions conducted at pH 5.7 had 60% less bioaccessible MeHg than digestions conducted at pH 6.4. In conclusion, both digestive and absorptive processes seem to have a role in reduced MeHg bioavailability, thus suggesting that ingested MeHg is less than 100% bioavailable. More accurate estimates of MeHg bioavailability can help properly balance the toxicological risks and the nutritional benefits of consuming seafood, though additional research is needed before use in risk assessment.
MERCURY TRANSFER ACROSS THE HUMAN PLACENTA: FROM KINETICS TO GENETICS
Mercury is well known to cross the placenta, to accumulate in placental tissue, and to pass onto fetal blood and fetal organs. An active transport across the placenta has been assumed. The underlying mechanisms however were virtually unknown. The genetic factors modulating placental toxicokinetics remain unclear just as well. The placenta is a unique organ. The tissue phenotype, i.e., placental protein level and function, can easily be determined. The term placenta is non-invasively available and, in addition, primary cells can be isolated therefrom.
In a recent project on mercury toxicokinetics in healthy human term placentas, candidate proteins putatively involved in mercury uptake, metabolisation and efflux were examined. Expression, localization and function of overall 27 proteins were determined in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. To prove involvement of the proteins in placental mercury metabolism, we used small interfering RNA (siRNA) and exposed cells to methylmercury. Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy.
We found two amino acid transporter subunits (LAT1, rBAT) and one ABC transporter (MRP1) to be involved in mercury toxicokinetics of trophoblast cells. According to our data, a model could be deduced which can explain why mercury is efficiently transported towards the fetal side: It uses LAT1 (probably also LAT2) and rBAT dimerized to another light chain than b0,+ at the apical side of the syncytiotrophoblastto enter trophoblast cells. Intracellular mercury dissociates from cysteine, binds to glutathioneand is therefore no longer a substrate of amino acid transporters. Mercury conjugated to glutathione is effluxed by MRP1. We made further important observations. Human trophoblast cells show strong variation in mercury accumulation capacity and in protein expression levels. It is reasonable that abundance and activity of the involved transporters determine mercury transfer rates across the syncytiotrophoblast. This raises the question on the regulatory factors behind. In our search on genetic markers, we found some evidence for sequence variants including one ABCC1 polymorphism to be related to placental mercury toxicokinetics.
UNCOVERING THE GUT MICROBIOME MACHINERY BEHIND MERCURY TRANSFORMATION
Fish is an important source of nutrients essential for functions including brain growth and development. However, contaminants such as methylmercury (MeHg) bioaccumulate in fish, and biomagnify along the food chain resulting in higher concentrations among predatory fish. Due to increasing anthropogenic activities, mercury (Hg) has been remobilized in the environment leading to increases in fish Hg concentrations. Epidemiological studies show great variation in Hg uptake between different human populations but also between individuals within a population. Although, the genetic makeup of an individual plays a role in this variation, the gut microbiota of individuals can also play an essential role in the transformation of Hg. Study of the gut microbiome is a growing field of research that has shown links between the gut microbiota to its hosts physiology, endocrinology and its immune system. Numerous studies have shown link between GI functions and gut microbial community structure including its ability to demethylate MeHg. However, little is known about the roles of gut microorganisms, if any, on mercury transformations. Our goal is to test the hypotheses that certain taxa of bacteria are responsible for MeHg transformations and components of the diet can mediate shifts in gut microbiome that can affect MeHg transformations. Using a series of batch experiments, we evaluated the effects of diet (by altering relative abundances of carbohydrate, fiber, lipid, or protein) on the gut microbial community structure of several individuals. We tracked mercury methylation and demethylation rates for each diet and conducted high throughput sequencing of the 16S rRNA gene fragments to determine microbial community structure. For one individual, we observed 100% decrease in MeHg concentration in a protein-rich medium. Our data suggest that two selected taxa could be responsible for microbial mediated mercury demethylation in the gut environment. Both have not been reported to have mercury demethylation capabilities and the mechanism remains unknown. This study is an important first step in understanding the mechanism behind mercury transformation in the gut microbiome.
MERCURY SPECIATION AND SUBCELLULAR DISTRIBUTION IN EXPERIMENTALLY-DOSED AND WILD BIRDS
Many bird species are exposed to methylmercury (MeHg) at levels shown to cause sub-lethal effects, including impaired physiology, behavior, and reproduction. However, the risk for negative health effects of MeHg can vary among species and with age. Birds can assimilate dietary MeHg in liver tissues, however, considerable variability in concentrations has been observed among species and individuals. Differences in MeHg toxicokinetics may play a role in the variability observed in MeHg assimilation and risk. Recent studies investigating how metals are distributed at a subcellular level have categorized subcellular fractions into metal-sensitive fractions: heat-denatured proteins (HDP), mitochondria, and microsomes and lysosomes, and detoxified metal fractions: heat-stable proteins (HSP) and granule-like fraction. A greater proportion of Hg located in metal-sensitive fractions may indicate an increased health risk. The objective of this study was to increase the understanding of MeHg toxicokinetics in birds by determining liver mercury (Hg) speciation and subcellular distribution in two avian species and across developmental time points. We used MeHg egg injection of a model avian species, White Leghorn Chicken (Gallus domesticus), to investigate liver Hg speciation and subcellular distribution among day 19 embryos, and day 1 and 7 hatchlings. We compared these results with those from maternally deposited Hg in wild embryonic Ring-billed Gulls (Larus delawarensis). THg in liver was mostly MeHg for all species and time points, indicating little MeHg demethylation in embryos and hatchlings. THg concentrations in chicken livers ranged from 1.8 – 11.8 µg/g dw, with lower concentrations seen in embryos. Embryonic Ring-billed Gull liver tissues had much lower THg concentrations, from 0.08 – 0.17 µg/g dw. Chicken and Ring-billed Gull liver tissues were also subjected to a subcellular partitioning procedure using differential centrifugation, NaOH digestion, and heat denaturation. Subcellular fractions were analyzed for THg and the proportion of Hg in each fraction was determined, relative to the total Hg burden. The proportion of Hg in subcellular fractions differed little among time points for chicken liver, with embryos having lower Hg in HSP. Subcellular distribution also differed little between embryonic chicken and Ring-billed Gulls, with gulls having greater Hg in HSP. All species and time points had a greater proportion of Hg in the metal-sensitive fractions, with HDP and mitochondria proportions combined ranging from 58.8 – 61.7%. These results indicate an increased health risk from MeHg exposure in embryos and hatchings. This study can improve understanding of MeHg toxicokinetics and how it relates to MeHg sensitivity in birds.
VARIABILITY IN METHYLMERCURY (MEHG) METABOLISM, ELIMINATION RATE, AND GUT MICROBIOME COMPOSITION IN HUMANS FOLLOWING FISH CONSUMPTION, THE MEHG METABOLISM AND ELIMINATION STATUS (MERMES) STUDY.
Concern about methylmercury (MeHg) exposure has resulted in fish consumption advisories. These advisories have been controversial and difficult to establish because of the highly variable outcomes of MeHg exposure seen across populations as well as between individuals. This variability may be due to individual and population differences in MeHg metabolism. In the human body, elimination of mercury (Hg) stemming from a MeHg exposure occurs slowly (kel ~ 0.01 day-1 or t1/2 ~70 days) and thus is a major determinant of the Hg body burden. Human MeHg elimination, as determined by a variety of methods, is highly variable ranging from t1/2=35 to >150 days. The mechanisms that control MeHg toxicokinetics in the human body remain poorly understood. Nonetheless, microbial demethylation of MeHg in the lumen of the gut is thought to play a role by producing inorganic Hg (I-Hg) that is readily excreted in feces. To shed light on the biological determinants controlling MeHg body burden we have examined MeHg elimination and metabolism in a cohort of 40 subjects. Subjects ate three tuna fish meals, each one week apart, followed by a 60-day elimination period where no fish or seafood was consumed. Longitudinal analysis of hair (collected on day 60) was performed for 202Hg and 34S by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to derive hair growth rate and determine Hg elimination rates. Fecal samples collected during the elimination period were analyzed for %I-Hg as a proxy for MeHg demethylation. Elimination rates were found to vary more than two-fold ranging from kel=0.0246-0.0103 day-1 (estimated t1/2= 28.2-67.3 days). The method also proved sensitive in detecting Hg exposures occurring prior to the fish meals. Across all subjects, faster elimination rates were seen to positively associate with the %I-Hg in feces, consistent with the notion that demethylation plays a role in MeHg metabolism and excretion. In parallel with our elimination studies, we sampled feces of our participants before and twice after tuna consumption for gut microbiome characterization. Bacterial abundance and diversity was determined via 16S rRNA sequencing and phylogenetic analysis. A pattern of differential abundance over the course of the fish meals can be seen in select taxa. Preliminary analyses, across the subjects showed select taxa associated with faster Hg eliminators than in the slower eliminators. Our results highlight the existence of individual variation in MeHg metabolism and elimination and point to a central role for MeHg demethylation in this variation. (NIEHS P30ES001247, R21ES024859).
TOXICOKINETICS OF METHYLMERCURY IN NORTH ATLANTIC PILOT WHALES (GLOBICEPHALA MELAS)
Methylmercury (MeHg) is a neurotoxin that biomagnifies in food chains. High levels of MeHg have been observed in both marine mammals and humans. Although the adverse health effects of MeHg are well-documented, the toxicokinetics of MeHg in mammals are not as well understood. The main objective of this study is to better understand MeHg metabolism in long-finned pilot whales (Globicephala melas). To do this, we constructed a toxicokinetic model parameterized by analytical measurements of total Hg, MeHg and stable Hg isotope in various organs (brain, heart, kidney, liver, muscle, placenta, and spleen) from seven individuals. We also investigated how physical characteristics like age, sex, and length affect metabolism. Results show the liver had the highest total mercury and MeHg concentrations (adult mean total Hg = 146.5 mg/g ww, MeHg = 7.3 mg/g ww) but also the lowest fraction of MeHg (adult mean percentage = 5.4%). The highest percentages of MeHg were found in the heart and muscle. Additionally, estimated age (range: 2-32 years) is strongly positively correlated with several organs total mercury concentration (R2 = 0.84 for brain, 0.58 for heart, 0.83 for kidney, 0.85 for liver, 0.79 for muscle) and MeHg concentrations (R2 = 0.71 for liver, 0.62 for muscle). The fraction of methylmercury, however, significantly decreases with age for brain (R2 = 0.91), heart (R2 = 0.61), kidney (R2 = 0.58), and liver (R2 = 0.61), suggesting that whales demethylation capability may improve with growth. Stable Hg isotope measurements in each organ will be presented to enhance understanding of potential mechanisms affecting metabolism and variability of in situ demethylation among whale individuals. The findings of this study will extend our understanding of how metabolism alters the internal body burden of MeHg and will shed light on sources of currently unexplained MeHg variability observed in marine mammals and human populations.